H+ transport by uncoupling protein (UCP-1) is dependent on a histidine pair, absent in UCP-2 and UCP-3.
UCP from brown adipose tissue of hamster (now UCP-1) expressed in Saccharomyces cerevisiae was used to examine the role of a conspicuous histidine pair H145 and H147 which is conserved among UCP-1 from various animals. Single and double mutants were generated by converting H145 and H147 into neutral residues (H145Q and H147N). As measured by fluorescence of dansyl-GTP binding, the level of expression of the mutant UCP was the same as wild-type (wt) in the isolated mitochondria. With the isolated and reconstituted UCP, transport of H+ and Cl- were measured. The fatty acid dependent H+ transport was reduced to about 10% in the single mutant H145Q and H147N and almost abolished in the double mutant, whereas Cl- transport into these vesicles was not affected as compared to wt. The possible involvement of the His pair in nucleotide binding and its pH dependence were examined by determining the KD and the kinetics for [14C]GTP and [14C]ADP binding. There were no marked changes in the affinity as well as in the binding and dissociation rates toward both these nucleotides in the mutant versus wt. Thus, the involvement in nucleotide binding can be excluded. The His pair is localized on the matrix side, probably at the entrance of the H+ translocation channel in UCP-1. It is absent in the recently discovered UCP-2, and therefore, UCP-2 might be predicted not to be a H+ transporter or to use a different mechanism. UCP-3 is deficient only in the equivalent H145 and thus can be predicted to still sustain a reduced H+ transport. The data support our contention that H+-dissociation side chains of UCP-1 are involved in H+ transport in cooperation with fatty acid carboxyl groups.
Bienengraeber
M
M
Institute of Physical Biochemistry, University of Munich, Germany.
Echtay
K S
KS
Klingenberg
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014475:Uncoupling Agents / Q000378:metabolism; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3
D002352:Carrier Proteins; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011522:Protons; D011994:Recombinant Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3; D006639:Histidine; D019204:GTP-Binding Proteins